Firefly luciferase assay substrate

PCMs were washed three times with 1xPBS and then harvested by Luciferase lysis buffer containing 20 mM Tris pH 7.4 and 0.1% Triton X-100. The enzymatic activity in the lysates was measured using a Lumat LB (Berthold) luminometer after addition of Firefly Luciferase assay substrate (E1501, Promega) or Renilla Luciferase assay substrate (E2820, Promega). Firefly Luciferase values were normalized to the corresponding Renilla luciferase activity to normalize for transfection efficiency and the values were expressed as fold activation over corresponding control conditions.

IL-6 promoter reporter constructs were a gift from Dr. Ernesto Canalis (Department of Research, Saint Francis Hospital and Medical Centre, Hartford, USA) with the permission of Dr. George Fey (University of Erlangen-Nuremberg, Erlangen, Germany). The constructs were transfected and cells were treated as described previously in “Cell Culture” section. Luciferase activity driven by the IL-6 promoter construct was measured by combining the lysate with firefly luciferase assay substrate (Promega, Madison, WI) according to manufacturer's protocol. Luciferase activity was normalized to respective firefly Renilla activity.

Luciferase assays were performed as described previously [30 (link)] with the following modifications. Subgenomic replicon RNA (5 μg of in vitro transcribed RNA) was electroporated into HeLa cells. The cells were incubated in normal growth media (DMEM/F12 supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, 5 mL/1 × 10⁶ cells) and 1 × 10⁵ cells were harvested and lysed using 100 μL of 1X cell culture lysis reagent (CCLR, Promega) at the indicated times post-electroporation. Luciferase activity was measured by adding equal volume of firefly luciferase assay substrate (Promega) to cell lysate and the reaction mixture was applied to a Junior LB 9509 luminometer (Berthold Technologies) to read relative light units (RLU) for 10 s. Relative light units (RLU) were then normalized based on the total protein concentration determined by Bio-Rad protein assay reagent (BioRad) for each sample.